The receptor for folic acid is an established tumor marker, showing elevated expression in many epithelial cancers, including cancers of the ovary, cervix, endometrium, kidney, brain and head and neck. When folic acid is covalently linked to another molecule or particle, it may still bind with high affinity (KD -1 0-9M) to the folate receptor (FR), but will lose all affinity for the reduced folate carrier (a transport protein that mediates folate uptake by many nonmalignant cells). Folate conjugates are, therefore, bound and internalized only by FR-expressing cells. Because of FR upregulation on cancer cells, folate ligation has been hypothesized to convert the vitamin into a molecular "Trojan Horse" that can facilitate targeting and delivery of attached therapeutic or imaging agents into malignant cells. While results from cell culture studies have been very encouraging, few quantitative data are available to permit an assessment of the therapeutic potential of folatemediated drug targeting in human patients. In our first two aims, we propose to first obtain this quantitative information. In the last two aims, we will test the therapeutic potential of the strategy in mouse tumor models. First, we will measure the in vivo recycling rate of FR in several relevant cancer models. Together with published data on the levels of FR expression in various human cancers, this recycling information should enable a more quantitative estimate of the total uptake and delivery capacity of the folate-mediated targeting pathway. Second, we will address how the size of a folate conjugate impacts its accessibility to cancer cells in vivo. Recent data indicate that the ability of folate conjugates to bind to and decorate cells throughout a tumor mass may be limited by molecular size. Quantitative data on this matter will be required to guide the design of folate-linked therapeutics. Third, we will synthesize and test folate-conjugated cytotoxic drugs for therapeutic efficacy in vivo. And finally, we will define the molecular and cellular bases of a novel folate-targeted immunotherapy that we have already shown can eradicate established tumors in mice without damaging normal tissues.